Telomerase is an essential ribonucleoprotein reverse transcriptase that uses an RNA subunit called TER as a template to synthesize telomeric DNA onto chromosome ends. Telomerase activity is required for genome integrity and to promote cell proliferation and hence understanding how telomerase is regulated can provide insight into fundamental mechanisms that impede carcinogenesis. Telomerase function is controlled at the chromosome terminus by telomere binding proteins. One key telomere protein is Protection of Telomeres (POT) 1. POT1 binds telomeric DNA in yeast and vertebrates via an oligonucleotide/oligosaccharide (OB-fold). Preliminary results from the Shippen lab indicate that the flowering plant Arabidopsis thaliana harbors two divergent TER genes and three divergent POT1 genes. Unexpectedly, POT1a and POT1b bind TER1 and TER2, respectively, instead of telomeric DNA. Moreover, POT1a and TER1 function as positive regulators of telomerase activity, while POT1b and TER2 negatively regulate the enzyme. Thus, duplication of TER and POT1 appears to have resulted in the evolution of a novel mode of telomerase regulation. The overarching goal of the proposed research is to elucidate the residues in POT1a responsible for TER1 interaction to help explain how POT1a evolved to bind telomerase RNA instead of telomeric DNA and to promote telomerase enzyme function in vivo. The central hypothesis driving this research is that POT1a and TER1 are co-evolving and that the rapid evolution observed in TER1 is reflected in POT1a at specific amino acid residues. A molecular evolutionary approach is proposed to achieve the following specific aims. Aim 1 is to investigate when the POT1 and TER duplications occurred to look for evidence of co-evolution. Using PCR and Southern blots, the presence or absence of multiple POT1 and TER sequences will be determined for 15 species closely related to A. thaliana. The presence/absence of multiple gene copies will be mapped onto the organismal phylogeny of these species to determine whether the two genes co-diversified, an indication that they likely co- evolved. Aim 2 is to define residues in POT1a OB1 important for TER1 interaction and POT1a function in vivo. Tests for elevated levels of non-synonymous mutation in the gene sequence of POT1a will be used to identify amino acid sites likely shaped by adaptive evolution. Aim 3 is to employ a cross-species genetic complementation assay to characterize POT1a-TER1 evolution. POT1a copies from six species with varying degrees of relatedness to A. thaliana will be tested for their ability to complement the A. thaliana POT1a mutant. Aim 4 is to develop an evolutionarily-informed functional model for POT1a-TER interaction. Residues shaped by adaptive evolution will be tested by site-directed mutagenesis followed by complementation to determine how changes to these sites affect the ability of POT1a to bind TER1. Results of this study will provide new insight into the evolution of the POT1a-TER1 interaction and form the basis of a dynamic, first-of- its kind model of POT1-nucleic acid interactions. PUBLIC HEALTH RELEVANCE: Telomeres are essential for genome integrity and as a consequence, understanding how the telomere complex safeguards genome stability will be crucial for elucidating the fundamental mechanisms that regulate cell proliferation and impede carcinogenesis. Studies in model organisms have established the paradigms for human telomere biology, and continue to uncover novel telomere components and regulatory mechanisms. In this tradition, we will exploit the genetic tractability of Arabidopsis, its detailed molecular phylogeny and the multitude of telomere-related assays developed in this system to investigate how the telomerase RNP complex is evolving in multicellular organisms.